A. CD19
CD19 (B lymphocyte surface antigen B4, Genbank accession number M28170) is a 95 kDa type I transmembrane glycoprotein of the immunoglobulin superfamily (Stamenkovic, I. et al. (1988) “CD19, The Earliest Differentiation Antigen Of The B Cell Lineage, Bears Three Extracellular Immunoglobulin-Like Domains And An Epstein-Barr Virus-Related Cytoplasmic Tail,” J. Exper. Med. 168(3):1205-1210; Tedder, T. F. et al. (1989) “Isolation Of cDNAs Encoding The CD19 Antigen Of Human And Mouse B Lymphocytes. A New Member Of The Immunoglobulin Superfamily,” J. Immunol. 143(2):712-717; Zhou, L. J. et al. (1991) “Structure And Domain Organization Of The CD19 Antigen Of Human, Mouse, And Guinea Pig B Lymphocytes. Conservation Of The Extensive Cytoplasmic Domain,” J. Immunol. 147(4):1424-1432). CD19 is expressed on follicular dendritic cells and on all B cells from early pre-B cells at the time of heavy chain rearrangement up to the plasma cell stage, when CD19 expression is down regulated. CD19 is not expressed on hematopoietic stem cells or on B cells before the pro-B cell stage (Sato et al. (1995) “The CD19 Signal Transduction Molecule Is A Response Regulator Of B-Lymphocyte Differentiation,” Proc. Natl. Acad. Sci. (U.S.A.) 92:11558-62; Loken et al. (1987) “Flow Cytometric Analysis of Human Bone Marrow. II. Normal B Lymphocyte Development,” Blood, 70:1316-1324; Wang et al. (2012) “CD19: A Biomarker For B Cell Development, Lymphoma Diagnosis And Therapy,” Exp. Hematol. and Oncol. 1:36).
CD19 is a component of the B cell-receptor (BCR) complex, and is a positive regulator of B cell signaling that modulates the threshold for B cell activation and humoral immunity. CD19 interacts with CD21 (CR2, C3d fragment receptor) and CD81 through two extracellular C2-type Ig-like domains, forming together with CD225 the BCR complex. The intracellular domain of CD19 is involved in intracellular signaling cascades, primarily but not exclusively regulating signals downstream of the BCR and CD22 (Mei et al. (2012) “Rationale of Anti-CD19 Immunotherapy: An Option To Target Autoreactive Plasma Cells In Autoimmunity,” Arthritis Res and Ther. 14(Suppl. 5):S1; Wang et al. (2012) “CD19: A Biomarker For B Cell Development, Lymphoma Diagnosis And Therapy,” Exp. Hematol. and Oncol. 1:36); Del Nagro et al. (2005) “CD19 Function in Central and Peripheral B-Cell Development,” Immunologic Res. 31:119-131).
Several properties of CD19 suggest its possible potential as a target for immunotherapy. CD19 is one of the most ubiquitously expressed antigens in the B cell lineage and is expressed on >95% of B cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's Lymphoma (NHL) (Wilson, K. et al. (2010) “Flow Minimal Residual Disease Monitoring Of Candidate Leukemic Stem Cells Defined By The Immunophenotype, CD34+CD38lowCD19+ In B-Lineage Childhood Acute Lymphoblastic Leukemia,” Haematologica 95(4):679-683; Maloney, D. G. et al. (1997) “IDEC-C2B8 (Rituximab) Anti-CD20 Monoclonal Antibody Therapy In Patients With Relapsed Low-Grade Non-Hodgkin's Lymphoma,” Blood 90(6):2188-2195; Vose, J. M. (1998) “Current Approaches To The Management Of Non-Hodgkin's Lymphoma,” Semin. Oncol. 25(4):483-491; Nagorsen, D. et al. (2012) “Blinatumomab: A Historical Perspective,” Pharmacol. Ther. 136(3):334-342; Topp, M. S. et al. (2011) “Targeted Therapy With The T-Cell-Engaging Antibody Blinatumomab Of Chemotherapy-Refractory Minimal Residual Disease In B-Lineage Acute Lymphoblastic Leukemia Patients Results In High Response Rate And Prolonged Leukemia-Free Survival,” J. Clin Oncol. 2011 Jun. 20; 29(18):2493-2498; Nadler, et al. (1983) “B4, A Human B Lymphocyte-Associated Antigen Expressed On Normal, Mitogen-Activated, And Malignant B Lymphocytes.” J. Immunol.; 131:244-250; Ginaldi et al. (1998) “Levels Of Expression Of CD19 And CD20 In Chronic B Cell Leukaemias,” J. Clin. Pathol. 51:364-369; Anderson et al. (1984) “Expression of Human B Cell-Associated Antigens on Leukemias and Lymphomas: A Model of Human B Cell Differentiation,” Blood 63:1424-1433). CD19 is expressed on few if any other cell types and is also not expressed on terminally differentiated plasma cells, which thus may be spared by CD19-directed therapies. CD19 is not shed into the circulation, and can rapidly internalize (Ma et al. (2002) “Radioimmunotherapy For Model B Cell Malignancies Using 90Y-Labeled Anti-CD19 And Anti-CD20 Monoclonal Antibodies,” Leukemia 16:60-66; Raufi et al. (2013) “Targeting CD19 in B-cell lymphoma: emerging role of SAR3419,” Cancer Management Res 3:225-233). Notably, CD19 expression is maintained on B cell lymphomas that become resistant to anti-CD20 therapy (Davis et al. (1999) “Therapy of B-cell Lymphoma With Anti-CD20 Antibodies Can Result In The Loss Of CD20 Antigen Expression.” Clin Cancer Res, 5:611-615, 1999). CD19 has also been suggested as a target to treat autoimmune diseases (Tedder (2009) “CD19: A Promising B Cell Target For Rheumatoid Arthritis,” Nat. Rev. Rheumatol. 5:572-577).
B cell malignancies represent a heterogeneous group of disorders with widely varying characteristics and clinical behavior. Historically, most patients with symptomatic disease received a combination of non-cross-reactive genotoxic agents with the intent of achieving a durable remission and, in some cases, a cure. Although effective, many traditional regimens are also associated with considerable acute and long-term toxicities (see, e.g., Stock, W. et al. (2013) “Dose Intensification Of Daunorubicin And Cytarabine During Treatment Of Adult Acute Lymphoblastic Leukemia: Results Of Cancer And Leukemia Group B Study 19802,” Cancer. 2013 January 1; 119(1):90-98). The anti-CD20 monoclonal antibody rituximab is used for the treatment of many B cell disorders, where it may be employed in combination with “standard of care” agents or employed as a single agent (Fowler et al. (2013) “Developing Novel Strategies to Target B-Cell Malignancies,” Targeted Pathways, B-Cell Lymphoma in ASCO Educational Book, pp. 366-372; Bargou, R. et al. (2008) “Tumor Regression In Cancer Patients By Very Low Doses Of A T Cell-Engaging Antibody,” Science 321(5891):974-977; Thomas, D. A. et al. (2010) “Chemoimmunotherapy With A Modified Hyper-CVAD And Rituximab Regimen Improves Outcome In De Novo Philadelphia Chromosome-Negative Precursor B-Lineage Acute Lymphoblastic Leukemia,” J. Clin. Oncol. 28(24):3880-3889). Despite encouraging clinical results, retreatment of patients with indolent lymphomas with single agent rituximab has been associated with a response rate of only 40%, suggesting that resistance may occur in malignant B cells as a response to prolonged exposure to rituximab (Smith (2003) “Rituximab (Monoclonal Anti-CD20 Antibody): Mechanisms Of Action And Resistance.” Oncogene. 22:7359-68; Davis et al. (1999) “Therapy of B-cell Lymphoma With Anti-CD20 Antibodies Can Result In The Loss Of CD20 Antigen Expression,” Clin Cancer Res, 5:611-615, 1999; Gabrilovich, D. et al. (2003) “Tumor Escape From Immune Response: Mechanisms And Targets Of Activity,” Curr. Drug Targets 4(7):525-536). Rituximab-resistant cell lines generated in vitro have been reported to display cross-resistance against multiple chemotherapeutic agents (Czuczman et al. (2008) “Acquirement Of Rituximab Resistance In Lymphoma Cell Lines Is Associated With Both Global CD20 Gene And Protein Down-Regulation Regulated At The Pretranscriptional And Post-transcriptional Levels,” Clin Cancer Res. 14:1561-1570; Olejniczak et al. (2008) “Acquired Resistance To Rituximab Is Associated With Chemotherapy Resistance Resulting From Decreased Bax And Bak Expression,” Clin Cancer Res. 14:1550-1560).
Additional B cell lymphoma surface antigens targeted by therapeutic antibodies include CD19 (Hoelzer (2013) “Targeted Therapy With Monoclonal Antibodies In Acute Lymphoblasitic Leukemia, Curr. Opin. Oncol. 25:701-706; Hammer (2012) “CD19 As An Attractive Target For Antibody-Based Therapy,” mAbs 4:571-577). However, despite the potent anti-lymphoma activities associated with complex internalization and intracellular release of free drug were reported for various anti-CD19 antibodies bi-specific antibodies having an anti-CD19 binding portion or antibody drug conjugates (ADCs) the anti-tumor effects of these anti-CD19 antibodies or ADCs were variable, suggesting that antibody specific properties, such as epitope binding, the ability to induce CD19 oligomerization or differences in intracellular signaling affect their potencies (Du et al. (2008) “Differential Cellular Internalization Of Anti-CD19 And-CD22 Immunotoxins Results In Different Cytotoxic Activity,” Cancer Res. 68:6300-6305; Press et al. (1994) “Retention Of B-Cell-Specific Monoclonal Antibodies By Human Lymphoma Cells. Blood,” 83: 1390-1397; Szatrowski et al. (2003) “Lineage Specific Treatment Of Adult Patients With Acute Lymphoblastic Leukemia In First Remission With Anti-B4-Blocked Ricin Or High-Dose Cytarabine: Cancer And Leukemia Group B Study 9311,” Cancer 97:1471-1480; Frankel et al. (2013) “Targeting T Cells To Tumor Cells Using Bispecific Antibodies,” Curr. Opin. Chem. Biol. 17:385-392).
Thus, despite improvements in survival for many, the majority of patients suffering from B cell malignancies continue to relapse following standard chemoimmunotherapy and more than 15,000 patients still die annually in the United States from B cell cancers. Accordingly, treatments with new non-cross-resistant compounds are needed to further improve patient survival.
B. CD3
CD3 is a T cell co-receptor composed of four distinct chains (Wucherpfennig, K. W. et al. (2010) “Structural Biology Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling,” Cold Spring Harb. Perspect. Biol. 2(4):a005140; pages 1-14; Chetty, R. et al. (1994) “CD3: Structure, Function, And Role Of Immunostaining In Clinical Practice,” J. Pathol. 173(4):303-307; Guy, C. S. et al. (2009) “Organization Of Proximal Signal Initiation At The TCR: CD3 Complex,” Immunol. Rev. 232(1):7-21).
In mammals, the complex contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) in order to generate an activation signal in T lymphocytes (Smith-Garvin, J. E. et al. (2009) “T Cell Activation,” Annu. Rev. Immunol. 27:591-619). In the absence of CD3, TCRs do not assemble properly and are degraded (Thomas, S. et al. (2010) “Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer,” Immunology 129(2):170-177). CD3 is found bound to the membranes of all mature T cells, and in virtually no other cell type (see, Janeway, C. A. et al. (2005) In: IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH AND DISEASE,” 6th ed. Garland Science Publishing, NY, pp. 214-216; Sun, Z. J. et al. (2001) “Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3ε:γ Heterodimer,” Cell 105(7):913-923; Kuhns, M. S. et al. (2006) “Deconstructing The Form And Function Of The TCR/CD3 Complex,” Immunity. 2006 February; 24(2):133-139).
The invariant CD3ε signaling component of the T cell receptor (TCR) complex on T cells, has been used as a target to force the formation of an immunological synapse between T cells and tumor cells. Co-engagement of CD3 and the tumor antigen activates the T cells, triggering lysis of tumor cells expressing the tumor antigen (Baeuerle et al. (2011) “Bispecific T Cell Engager For Cancer Therapy,” In: BISPECIFIC ANTIBODIES, Kontermann, R. E. (Ed.) Springer-Verlag; 2011:273-287). This approach allows bi-specific antibodies to interact globally with the T cell compartment with high specificity for tumor cells and is widely applicable to a broad array of cell-surface tumor antigens.
C. Antibodies and Other Binding Molecules
Antibodies are immunoglobulin molecules capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also mutants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen recognition site of the required specificity, humanized antibodies, and chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
The ability of an intact, unmodified antibody (e.g., an IgG) to bind an epitope of an antigen depends upon the presence of Variable Domains on the immunoglobulin light and heavy chains (i.e., the VL and VH Domains, respectively). Interaction of an antibody light chain and an antibody heavy chain and, in particular, interaction of its VL and VH Domains forms one of the epitope binding sites of the antibody. In contrast, the scFv construct comprises a VL and VH Domain of an antibody contained in a single polypeptide chain wherein the Domains are separated by a flexible linker of sufficient length to allow self-assembly of the two Domains into a functional epitope binding site. Where self-assembly of the VL and VH Domains is rendered impossible due to a linker of insufficient length (less than about 12 amino acid residues), two of the scFv constructs interact with one another other to form a bivalent molecule in which the VL of one chain associates with the VH of the other (reviewed in Marvin et al. (2005) “Recombinant Approaches To IgG-Like Bispecific Antibodies,” Acta Pharmacol. Sin. 26:649-658).
In addition to their known uses in diagnostics, antibodies have been shown to be useful as therapeutic agents. The last few decades have seen a revival of interest in the therapeutic potential of antibodies, and antibodies have become one of the leading classes of biotechnology-derived drugs (Chan, C. E. et al. (2009) “The Use Of Antibodies In The Treatment Of Infectious Diseases,” Singapore Med. J. 50(7):663-666). Nearly 200 antibody-based drugs have been approved for use or are under development.
Natural antibodies are capable of binding to only one epitope species (i.e., mono-specific), although they can bind multiple copies of that species (i.e., exhibiting bi-valency or multi-valency). A wide variety of recombinant bi-specific antibody formats have been developed (see, e.g., PCT Publication Nos. WO 2008/003116, WO 2009/132876, WO 2008/003103, WO 2007/146968), most of which use linker peptides either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scFv. Typically, such approaches involve compromises and trade-offs. For example, PCT Publications Nos. WO 2013/174873, WO 2011/133886 and WO 2010/136172 disclose that the use of linkers may cause problems in therapeutic settings, and teaches a tri-specific antibody in which the CL and CH1 Domains are switched from their respective natural positions and the VL and VH Domains have been diversified (WO 2008/027236; WO 2010/108127) to allow them to bind to more than one antigen. Thus, the molecules disclosed in these documents trade binding specificity for the ability to bind additional antigen species. PCT Publications Nos. WO 2013/163427 and WO 2013/119903 disclose modifying the CH2 Domain to contain a fusion protein adduct comprising a binding domain. The document notes that the CH2 Domain likely plays only a minimal role in mediating effector function. PCT Publications Nos. WO 2010/028797, WO2010028796 and WO 2010/028795 disclose recombinant antibodies whose Fc Regions have been replaced with additional VL and VH Domains, so as to form tri-valent binding molecules. PCT Publications Nos. WO 2003/025018 and WO2003012069 disclose recombinant diabodies whose individual chains contain scFv domains. PCT Publications No. WO 2013/006544 discloses multi-valent Fab molecules that are synthesized as a single polypeptide chain and then subjected to proteolysis to yield heterodimeric structures. Thus, the molecules disclosed in these documents trade all or some of the capability of mediating effector function for the ability to bind additional antigen species. PCT Publications Nos. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2007/075270, WO 1998/002463, WO 1992/022583 and WO 1991/003493 disclose adding additional Binding Domains or functional groups to an antibody or an antibody portion (e.g., adding a diabody to the antibody's light chain, or adding additional VL and VH Domains to the antibody's light and heavy chains, or adding a heterologous fusion protein or chaining multiple Fab Domains to one another). Thus, the molecules disclosed in these documents trade native antibody structure for the ability to bind additional antigen species.
The art has additionally noted the capability to produce diabodies that differ from such natural antibodies in being capable of binding two or more different epitope species (i.e., exhibiting bi-specificity or multispecificity in addition to bi-valency or multi-valency) (see, e.g., Holliger et al. (1993) “‘Diabodies’: Small Bivalent And Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci. (U.S.A.) 90:6444-6448; US 2004/0058400 (Hollinger et al.); US 2004/0220388 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672; WO 02/02781 (Mertens et al.); Olafsen, T. et al. (2004) “Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications,” Protein Eng Des Sel. 17(1):21-27; Wu, A. et al. (2001) “Multimerization Of A Chimeric Anti-CD20 Single Chain Fv-Fv Fusion Protein Is Mediated Through Variable Domain Exchange,” Protein Engineering 14(2):1025-1033; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588; Baeuerle, P. A. et al. (2009) “Bispecific T-Cell Engaging Antibodies For Cancer Therapy,” Cancer Res. 69(12):4941-4944).
The design of a diabody is based on the single chain variable region fragments (scFv). Such molecules are made by linking light and/or heavy chain variable regions by using a short linking peptide. Bird et al. (1988) (“Single-Chain Antigen-Binding Proteins,” Science 242:423-426) describes example of linking peptides which bridge approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used (Bird et al. (1988) “Single-Chain Antigen-Binding Proteins,” Science 242:423-426). Linkers can in turn be modified for additional functions, such as attachment of drugs or attachment to solid supports. The single chain variants can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be made by routine manipulations such as ligation of polynucleotides. The resultant scFv can be isolated using standard protein purification techniques known in the art.
U.S. Pat. No. 7,585,952 and United States Patent Publication No. 2010-0173978 concern scFv molecules that are immunospecific for ErbB2. Bi-specific T cell engagers (“BITES”), a type of scFv molecule has been described (WO 05/061547; Baeuerle, P et al. (2008) “BiTE: A New Class Of Antibodies That Recruit T Cells,” Drugs of the Future 33: 137-147; Bargou, et al. 2008) “Tumor Regression in Cancer Patients by Very Low Doses of a T Cell-Engaging Antibody,” Science 321: 974-977). Such molecules are composed of a single polypeptide chain molecule having two antigen-binding domains, one of which immunospecifically binds to a CD3 epitope and the second of which immunospecifically binds to an antigen present on the surface of a target cell.
Bi-specific molecules that target both CD19 and CD3 have been described. Blinatumomab, a bi-specific scFv-CD19×CD3 BiTE is in clinical trials and is reported to have an affinity for CD3 of ˜100 nM, and an affinity for CD19 of ˜1 nM. Blinatumomab exhibits an EC50 for target cell lysis in vitro of ˜10-100 pg/ml (Frankel et al. (2013) “Targeting T Cells To Tumor Cells Using Bispecific Antibodies,” Curr. Opin. Chem. Biol. 17:385-392). A bi-specific CD19×CD3 dual affinity retargeting (DART) has also been developed (Moore et al. (2011) “Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma,” Blood 117(17):4542-4551). Compared with the bi-specific scFv-CD19×CD3 BiTE, this molecule exhibited similar binding affinities but had a lower EC50 for target cell lysis in vitro of ˜0.5 to 5 pg/ml and showed consistently higher levels of maximum lysis AFM11, a CD19×CD3 a bi-specific Tandab has also been described. AFM11 is reported to have an EC50 for target cell lysis in vitro of ˜0.5 to 5 pg/ml (Zhukovsky et al. (2013) “A CD19/CD3 Bispecific Tandab, AFM11, Recruits T Cells To Potently and Safely Kill CD19+ Tumor Cells,” J Clin Oncol 31, 2013 (suppl; abstr 3068)). None of these constructs include an Fc domain.
Notwithstanding such success, the production of stable, functional heterodimeric, non-mono-specific diabodies can be further optimized by the careful consideration and placement of cysteine residues in one or more of the employed polypeptide chains. Such optimized diabodies can be produced in higher yield and with greater activity than non-optimized diabodies. The present invention is thus directed to the problem of providing polypeptides that are particularly designed and optimized to form heterodimeric diabodies. The invention solves this problem through the provision of exemplary, optimized CD19×CD3 diabodies.